Histone deacetylases and the nuclear receptor corepressor regulate lytic-latent switch gene 50 in murine gammaherpesvirus 68-infected macrophages

Abstract

Gammaherpesviruses are important oncogenic pathogens that transit between lytic and latent life cycles. Silencing the lytic gene expression program enables the establishment of latency and a lifelong chronic infection of the host. In murine gammaherpesvirus 68 (MHV68, γHV68), essential lytic switch gene 50 controls the interchange between lytic and latent gene expression programs. However, negative regulators of gene 50 expression remain largely undefined. We report that the MHV68 lytic cycle is silenced in infected macrophages but not fibroblasts and that histone deacetylases (HDACs) mediate silencing. The HDAC inhibitor trichostatin A (TSA) acts on the gene 50 promoter to induce lytic replication of MHV68. HDAC3, HDAC4, and the nuclear receptor corepressor (NCoR) are required for efficient silencing of gene 50 expression. NCoR is critical for transcriptional repression of cellular genes by unliganded nuclear receptors. Retinoic acid, a known ligand for the NCoR complex, derepresses gene 50 expression and enhances MHV68 lytic replication. Moreover, HDAC3, HDAC4, and NCoR act on the gene 50 promoter and are recruited to this promoter in a retinoic acid-responsive manner. We provide the first example of NCoR-mediated, HDAC-dependent regulation of viral gene expression.

FIG. 1.

TSA increases the number of cells expressing MHV68 lytic proteins and increases MHV68 replication in macrophage cultures. (A) Infected RAW264.7 cells (MOI = 10) treated with DMSO or 130 nM TSA and examined by immunofluorescence for MHV68 lytic protein expression at 24 hpi (one of three experiments shown); (B and C) viral titer from RAW264.7 cells (mean ± standard error of the mean [SEM], 3 to 4 experiments) (B) and primary macrophages (BMmφ) (mean ± SEM, 3 to 5 experiments) (C) treated as described for panel A; (D) immunofluorescence of MEFs treated as described for panel A (the results of one of three experiments are shown); (E and F) viral titer from MEFs infected at an MOI of 10 (E) or an MOI of 0.05 (F) and treated as described for panel A (panel E, mean ± SEM, 5 experiments; panel F, mean ± SEM, 4 experiments). Statistical analyses were done by Student's t test.

FIG. 2.

TSA increases the frequency of macrophage cells that support MHV68 lytic replication. (A) Limiting dilutions of infected RAW264.7 cells (MOI = 10) plated onto MEF monolayers in the presence of DMSO or 130 nM TSA. Parallel samples were mechanically disrupted to kill the cells before they were plated. The statistical difference between TSA and DMSO is P < 0.05 (mean ± SEM, 3 experiments). (B) Limiting dilutions of MHV68 plated onto MEFs in the presence of DMSO or 130 nM TSA (mean ± SEM, 3 experiments). Dashed lines indicate the 63.2% Poisson distribution line used to calculate frequencies. Statistical analysis was done by paired t test.

FIG. 3.

HDAC inhibitors stimulate expression of gene 50 in macrophages. Infected cells (MOI = 10) were treated with HDAC inhibitor, and viral transcript levels were examined at 12 hpi. (A to C) Gene 50 and gene 73 transcript levels in RAW264.7 cells (mean ± SEM, 6 experiments) (A), primary macrophages (mean ± SEM, 5 to 7 experiments) (B), and MEF cells (mean ± SEM, 3 experiments) (C) treated with 130 nM TSA; (D) gene 50 transcript levels in RAW264.7 cells treated with 3 mM NaB (mean ± SEM, 3 experiments); (E) gene 50 transcript levels in RAW264.7 cells treated with 0.3 μM or 8 μM MS-275 (mean ± SEM, 4 experiments). Statistical analyses were done by Student's t test.

FIG. 4.

HDAC3 and HDAC4 silence gene 50 expression and MHV68 replication in macrophages. RAW264.7 cells were transfected with the indicated siRNA 24 h before they were infected with MHV68 (MOI = 10). (A and B) Gene 50 (A) and gene 73 (B) transcript levels at 12 hpi (mean ± SEM, 3 to 5 experiments); (C) viral titer (mean ± SEM, 2 to 4 experiments); (D) representative Western blots of siRNA knockdown and corresponding quantification of protein levels normalized to those for actin (mean ± SEM, 2 to 4 experiments); (E and F) gene 50 and gene 73 transcript levels in infected RAW264.7 cells (MOI = 10) transfected with 5 μg of pUB-HDAC3 (E; mean ± SEM, 3 experiments) or 4 μg of pUB-HDAC4 (F; mean ± SEM, 4 experiments); (G) representative Western blots of HDAC3 and HDAC4 expression in RAW264.7 treated as described for panels E and F. Statistical analyses were done by Student's t test. *, P < 0.05.

FIG. 5.

NCoR represses gene 50 expression and MHV68 replication in macrophages. (A and B) Gene 50 and gene 73 transcript levels measured at 12 hpi (mean ± SEM, 5 to 7 experiments); (C) viral titer (mean ± SEM, 3 to 4 experiments) from RAW264.7 cells transfected with the indicated siRNA 24 h before infection with MHV68 (MOI = 10); (D) representative Western blots of siRNA knockdown and corresponding quantification of protein levels normalized to those of actin (mean ± SEM, 3 to 5 experiments); (E and F) gene 50 and gene 73 transcript levels measured at 12 hpi (mean ± SEM, 4 experiments); (G) viral titer (mean ± SEM, 3 to 6 experiments) from RAW264.7 cells infected with MHV68 (MOI = 10) and treated with DMSO or 1 μM RA. Statistical analyses were done by Student's t test. *, P < 0.05; **, P < 0.01.

FIG. 6.

The gene 50 promoter is TSA responsive and NCoR, HDAC3, and HDAC4 are recruited to the promoter in an RA-dependent manner. (A and B) Uninfected primary macrophages were transfected with pGL2-410bp (A; mean ± SEM, 8 experiments) or pGL2-ORF57 (B; mean ± SEM, 5 experiments) and treated with DMSO or 130 nM TSA. (C) Viral titer from RAW264.7 cells cotransfected with 0.5 μg of pUB-Rta and 4 μg pUB-HDAC3 or empty vector (mean ± SEM, 3 to 5 experiments). (D and E) ChIP analysis of HDAC3, HDAC4, and NCoR recruitment to the 410-bp promoter and gene 57 promoter in infected primary macrophages (MOI = 10) at 12 hpi. Cells were treated at 2 hpi with DMSO, 130 nM TSA, or 1 μM RA (mean ± SEM, 3 to 4 experiments). (F) ChIP analysis of the 410-bp promoter and gene 57 promoter in infected MEF cells (MOI = 10) at 12 hpi using antibodies to HDAC3, HDAC4, and NCoR (mean ± SEM, 2 experiments). Statistical analyses were done by Student's t test (A to C) or the Mann-Whitney test (D to F). *, P < 0.05.